The aim of this research project is to understand how the positive control elements, D-serine deaminase activator protein and cyclic AMP-CAP complex, interact with the promoter-initiator region of the D-serine deaminase structural gene to promote its transcription. Activator, together with its ligand D-serine, is absolutely required for expression of the wild type D-serine deaminase operon: cyclic AMP-CAP is necessary for optimal expression. The activator has been partially purified and is assayed by its ability to effect in vitro D-serine deaminase synthesis. The promoter-initiator region has been located on a 2.2 kilobase pair restriction fragment and cloned on multicopy plasmids. We plan to purify the activator to homogeneity and characterize it, to sequence the wild-type and mutant promoter-initiator regions, and to characterize the interaction of the activator with the promoter-initiator. To enhance our yields of activator, we will attempt to further amplify the activator gene or its expression by several methods. In order to sequence the promoter-initiator it must be localized to a DNA fragment of 200 base pairs or less, and we will carry out further restriction analysis and cloning to obtain such a fragment. We then plan binding and nuclease protection analyses to determine the sites of interaction of activator and cyclic AMP-CAP with the promoter-initiator, and the effect of these interactions on the DNA.